The mechanism of copper uptake by tyrosinase from Bacillus megaterium
Margarita Kanteev, Mor Goldfeder, Michał Chojnacki, Noam Adir, and Ayelet Fishman [1]
Molecular Tour
Tyrosinase belongs to the type-3 copper enzyme family, containing a di-nuclear Cu center, CuA and CuB. It is mainly responsible for melanin production in a wide range of organisms. Although Cu ions are essential for the activity of tyrosinase, the mechanism of Cu uptake is still unclear.
We have recently determined the crystal structure of tyrosinase from Bacillus megaterium (TyrBm) and revealed that this enzyme has tighter binding of CuA in comparison to CuB. Using a rational design approach and structure-function investigations, we identified four residues at the second shell of the active site, having an impact on Cu uptake. Looking at the crystal structure of WT TyrBm, we have found that a major role of the highly conserved Asn205 residue is to stabilize the orientation of the His204 imidazole ring in the binding site, thereby promoting the correct coordination of CuB. Substitution of Asn205 to Ala or Asp residues resulted in significant reduction in copper uptake and enzymatic activity. In the crystal structure of variant N205D, residue Asp205 is turned towards Arg209 and therefore cannot form a polar bond with His204.
Furthermore, Phe197, Met61 and Met184, which are also located at the entrance to the active site, were found to play an important role in Cu uptake. Substitution of a bulky Phe197 to Ala, resulted in higher Cu uptake, while mutations at positions 61 and 184, had an opposite effect. In addition, it was found that these residues are also important for enhancing the diphenolase activity of the enzyme. Therefore, we can suggest that a Cu ion entering the active site (CuA) first associates with the methionine residues followed by its transfer to His60 in its flipped out conformation. Following Cu binding, His60 alters its conformation and transfers CuA into the active site in a paddle-like motion. We propose that CuB passes only through Asp205 and Phe197. The proposed pathway clarifies the previously reported observations of TyrBm’s tighter binding of CuA in comparison to CuB.
